The American Society for Microbiology (ASM) is submitting the following comments on the Environmental Protection Agency's (EPA) proposed rule for regulating microbial products of biotechnology under the Toxic Substances Control Act (TSCA) (59 Federal Register 45526-45585).

With a membership of 40,000, the ASM includes scientists from academe, industry and government, who are experts in molecular biology and genetics, environmental microbiology, microbial ecology, medical, agricultural and industrial microbiology. The ASM has a long record of involvement in the development of a coordinated framework for regulation of biotechnology products. The ASM has repeatedly advised government policymakers that biotechnology regulatory policy should be focused on organisms that pose a significant risk to the public health and the environment. The Society endorses the following statement from reports of the National Academy of Sciences: Although genetic modification by molecular methods may be more powerful and capable of producing a wider range of phenotypes, no conceptual distinction exists between genetic modification by classical methods or by molecular methods of modifying DNA...Assessment of an organism should be based on the nature of the organism and the environment into which it will be introduced, not on the methods by which it was produced.

Overall Evaluation of Proposed Rule

Unfortunately, the proposed EPA TSCA regulations for microbial products of biotechnology are not tailored to potential risks. Scientifically sound regulations for biotechnology should be risk based and would regulate phenotype--i.e. what the organism is, rather than process, i.e., how the organism is created. The proposed regulations circumvent scientific underpinnings related to risk. The EPA has proposed regulations under TSCA for biotechnology that represent a practical approach to a difficult problem. We recognize that the proposed regulations will provide a framework for the development and use of microbial products of biotechnology for environmental applications while protecting human health and environmental quality.

The proposed regulations represent an improvement over the current Pre-Manufacturing Notice (PMN) process. The PMN process was originally designed for chemicals, not microorganisms, and requires significant modification for application to biotechnology and the regulation of recombinant microorganisms. Additionally, the PMN system was designed for products ready for commercialization. Use of the PMN system for research and development (R&D) is cumbersome and difficult because the issues related to R&D activities differ greatly from those of full scale manufacturing. The current proposal for a two stage regulatory process--a TSCA Experimental Release Application (TERA) for regulated R&D activities as the first stage and a Microbial Commercial Activity Notice (MCAN) as the second stage--offers the potential to reduce significantly the regulatory burden for products that are still in the R&D phase.

The exemptions provided in the proposed TSCA regulations for microorganisms that are known to be safe to human health and the environment, based upon familiarity derived from extensive investigation and history of safe use, are a significant improvement over previously proposed EPA regulations. The exemption under the proposed TSCA regulations for natural (nonrecombinant) microorganisms, which may be deliberately released in applications such as bioaugmentation to enhance bioremediation, also is aimed at ensuring that the regulation of biotechnology under TSCA is not excessively burdensome. Further safety testing and regulation of these microorganisms and their uses are not needed.

Of particular importance in the proposed regulations is the explicit acceptance by the EPA that the NIH Guidelines for Recombinant DNA Research provide adequate oversight for contained R&D of recombinant DNA technology at institutions required to comply with the NIH Guidelines. Institutions that comply with the NIH Guidelines for contained R&D should be exempt from further regulation. This should include universities receiving federal funding, federal agencies and industry required to comply with the NIH Guidelines, e.g. by lease requirements or by institutional, municipal or state ordinance. Acceptance of the NIH Guidelines as the overriding oversight framework avoids unnecessary new regulations for contained research and, will allow the EPA to focus on products ready for commercialization and for introduction into the environment.

The EPA proposed regulation does not define "commercial purposes" but has signaled that its interpretation of "commercial purposes" will be very broad. We believe that the EPA should focus on commercial products that are intended for intentional introduction into the environment. The EPA should clearly define what is meant by commercial purposes to avoid undue regulation of research. There is no basis for EPA to consider all activities at a nonprofit institution to be commercial because one activity is commercial (page 45537). To do so would result in unnecessary, unwarranted and burdensome reporting requirements for academic research. Clear indices or criteria of commercial purpose should be presented to assist efforts to focus the regulations on commercial products and not on fundamental research. If criteria or clear indices cannot be agreed upon, it may be necessary to require that all intentional testing outside of contained structures be considered commercial.

In keeping with the NIH Guidelines, the EPA should expand its exemption of intergeneric recombinant DNA molecules to include recombinant DNA molecules that consist entirely of DNA segments from species that naturally exchange DNA by known physiological processes. This exemption of natural exchangers would make TSCA regulations of biotechnology consistent with the NIH Guidelines for Recombinant DNA. The NIH Director periodically prepares revised lists of such natural exchangers. For example, at present bacterial intergeneric exchanges between species of the genus Escherichia, the genus Shigella, the genus Salmonella--including Arizona, the genus Enterobacter, the genus Citrobacter--including Levinea, the genus Klebsiella--including K. oxytoca, the genus Erwinia, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas mendocina, Serratia marcescens, and Yersinia enterocolitica are listed in the NIH Guidelines as natural exchangers exempt from Institutional Biosafety Committee (IBC) registration. The ASM is prepared to assist the EPA in identifying a list of additional natural exchangers that would be exempt from regulation. There should be interagency cooperation between NIH and EPA and the EPA list should be forwarded to the NIH for incorporation into the NIH Guidelines.

Specific Comments

The following items are responses to requests for comments made on the pages indicated of the proposed rule as published in the Federal Register notice.

p. 45527C. Statutory Framework, b. Plants and animals are not subject to this proposed rule. In the last sentence of this section, EPA reserves the authority to regulate transgenic plants and animals under TSCA in the future. Since adequate regulations for transgenic plants and animals have already been promulgated by USDA, additional regulations by EPA under TSCA are not needed and will cause overlapping jurisdiction with other federal agencies.

p. 45527-82. Application of TSCA, Section 5. Since "any type of production" is considered 'manufactured,' all types of microbial growth, from test tubes to fermentors are 'covered.' This seems excessive unless exemptions are made for all activities in contained structures.

p. 45529e. Exemptions from the section 5 notification process. While EPA oversight at the R&D level may satisfy public concern, it is difficult to justify the time and expense for EPA TERAs. If IBCs are in place, a better alternative would be for the company/institution to forward the proposed experiment to the EPA with a 30-day period for review. This would constitute a combination of notification and review for those microorganisms unlikely to pose substantial or unreasonable risk.

p. 45529Requests for comment on approaches and alternatives: Although cumbersome, this assessment of whether or not reporting is needed seems reasonable, except for the concern about natural exchangers.

p. 45531b. State coordination. We believe it is a disincentive for R&D to be required to provide evidence for having notified state authorities of planned activities. This should only be applicable, if at all, to environmental, i.e. noncontained R&D.

p. 455322. MCAN submission and review process--a MCAN submission process. The prospective EPA "review that considers all (emphasis added) the reasonable ascertainable information on...effects" can be interpreted as open ended and subject to challenge both by submitters and opponents to biotechnology. It would be better to review only that information likely to be pertinent to the change in the phenotype of the organism.

p. 45534Figure 1. Tier 1 and Tier II should be added.

p. 455353. Requirements necessary for eligibility for exemptions from TERA reporting A. The contained structures exemption (ii) R&D not subject to another agency. An "authorized official" should include the possibility of using an IBC chair to prevent creating an additional layer of bureaucracy for an institution/business.

p. 45536 & 455375. Options for oversight of R&D activities. EPA claims a process of oversight commensurate with the level of risk, yet prejudices the science by assuming all genetically modified organisms are a greater risk than unmodified organisms. This assumption is unwarranted. The EPA should explicitly state that the proposed regulations under TSCA for microorganisms are not risk based.

p. 45536 & 45537Requests for comments on options for R&D activities and alternatives approach. The options for R&D appear flexible and reasonable for work in contained structures. The R&D option of specific alternative exemptions for low-risk field tests is endorsed.

We draw EPA's attention to the following publications on the field-testing of microorganisms: the USDA's ABRAC document (Guidelines for Research Involving Planned Introduction into the Environment of Genetically-Modified Organisms) in which risk categories were specifically addressed, OECD documents, and the book, "Microbial Ecology: Principles, Methods, and Applications," edited by M. A. Levin, R. J. Siedler, and M. Rogul, McGraw Hill, 1992.

p. 45536Since the TERA burden is structured to be minimal, the value of exemptions will rely primarily on the response time by EPA to exemption requests. Long delays in granting exemptions could negate the benefits of the exemption process and favor use of the TERA process which has mandated a 60-day review period (alternatively, 120 days if EPA extends the period for good reason).

p. 45539B. Exemption for Research in Contained Structures 2. Difficulties in ensuring that microorganisms used for R&D will not increase beyond small quantities. It is erroneous to presume that a non-replicating chemical cannot do localized or even wide-spread damage. It depends on the chemical's reactivity and potency. Conversely, it is erroneous to assume that self-sustaining or multiplying populations of microorganisms will do damage; they may actually be beneficial.

p. 45541It is erroneous to presume that the microorganisms used in small-scale field tests will establish themselves permanently in the environment. For example, even known deleterious organisms, such as plant pathogens are used annually in hundreds of tests without unreasonable adverse environmental effects.

p. 45544Request for comment on exemption from TERA reporting. We support the proposed exemptions for Bradyrhizobium japonicum and Rhizobium meliloti. Since these exemptions are limited to modifications originating from the same species, it is difficult to see how they could constitute an unreasonable risk. If it is necessary or desirable to have an EPA notification procedure it could be managed efficiently by IBCs or the PIs using electronic mail. We believe it would be prudent to limit recipient strains to be introduced into the environment to those that do not produce rhizobitoxine. This toxin, in combination with susceptible cultivars of soybeans, can cause chlorosis (yellows) and subsequent yield loss. If toxin-producing strains were to be used, a TERA should be filed.

p. 45545Request for comment on antibiotic-resistant traits in B. japonicim and R. Meliloti; The rationale for exemption is sound and reasonable. Potential exemptions should include other markers which have already been used in the environment without undesirable effects.

Request for comment on exemption for antibiotic-resistant markers. Antibiotic resistant markers are widely used and widely distributed in nature. Use of most such markers should pose no additional risk since they already occur in natural populations of microorganisms. However, certain antibiotic resistant markers for antibiotics critical in medical practice, such as methicillin and vancomycin should be avoided. The EPA should prepare a list of antibiotic resistant markers to avoid.

Several markers pose less of a risk than antibiotic resistance markers and should be exempt from TSCA regulation as an incentive for their use. These include lux and laczy reporter gene sequences that have a record of safe use as bioreporters and biomarkers.

p. 45545Request for comment on specific alternative exemption for R&D low-risk field tests. This potential exemption is reasonably based on the stated three primary criteria, but it is not clear how this would work in practice, since the TQI apparently must still notify EPA.

p. 45546Request for comment on criteria for tiered commercial use exemption: The criteria appear reasonable, except for the third, which is subject to interpretation of what constitues a history of safe use. A specific strain is often as significant as the species, as acknowledged below. The proposed recipients (page 45546-7) do not meet all the six evaluation criteria, including taxonomy. This is puzzling. If all the criteria have to be met for the exemption, to what degree do they have to be met? Perhaps no strains will qualify for exemption.

Request for comment on usefulness of introduced genetic material, size, limitation for exemption. This is a reasonable criterion except that EPA needs to recognize that all functions may not be known and predictable. This uncertainty can be reduced but not excluded.

p. 45548Request for comment on poorly mobilizable equals transfer frequency of 10-8. This definition seems reasonable and prudent. However, it can be argued that the trait or property is the significant issue, not the transfer frequency.

p. 45549Request for comment on standards for minimizing releases of microorganisms. These standards or numbers appear reasonable. If the submitter can show viability reduced to these levels, by any means, fermentor exhaust monitoring need not be required. (See comment on p. 45580)

p. 45550Request for comment on definition of microorganism. The definition is reasonable including members of plant groups. The pragmatic definition is: if an individual organism cannot be seen with the naked eye, it is a microorganism.

p. 45551Request for comment and appropriateness of intergeneric approach. Known uses of viruses for microorganisms subject to TSCA

No known uses are identified (beyond FIFRA)

The intergeneric approach for phages is awkward but doable.

Oversight of phage is reasonable.

p. 45551Taxonomic designation.The EPA should specify explicitly acceptable bases for designating the taxonomic identification of a microorganism. Modern classification systems are based on nucleic acid sequences, e.g. DNA or RNA analyses. Traditional identification is based on phenotypic characteristics, often using Bergey's manual as a reference. Taxonomic classifications are frequently changed. The EPA must describe and recommend a procedure for updating the taxonomic status of microorganisms that are to be regulated under TSCA.

Verification of taxonomic designation by a culture collection is potentially fraught with difficulty: cultures may be contaminated or they may display properties altered from those noted at the time of deposit. A reasonable number of tests must be performed so that the microorganism being submitted can be assigned to a taxon. However, the request for documentation of the genetic history back to the original isolate is onerous and time-consuming, and will not necessarily be useful.

p. 45551Request for comment on deposit in a culture collection. The proposed regulation is not clear as to when this would occur. Our understanding is that it would occur at the submission of an MCAN, not at the R&D stage. If this is the case, the requirement is satisfactory. If the requirement is at the R&D phase, it is not necessary and potentially onerous. Moreover, collections are not able to manage large numbers of experimental microorganisms for which there are no published reports or patents. Who will pay the cost of deposit and curating?

Comment on TSCA Inventory: Since only genera and species are mentioned, modifications that may be of significance and differ from those represented on the inventory may not be adequately reviewed.

p. 45554

5. Proposed Changes for Substantiation of CBI Claims. EPA is considering requesting up front substantiation of CBI claims for all TERA submissions. The proposed changes in this area are burdensome for R&D submitters and are not needed. This proposed change would require submission of CBI at an earlier stage of the process than required in current regulations. Once a product or process proceeds past R&D towards commercialization and a MCAN is submitted, CBI substantiation to EPA would be appropriate. However, it is not clear how non-profit institutions would proceed if they choose to submit a MCAN.

p. 45555 F.

Section 8(e) Reporting Requirements. These requirements remain problematic primarily because they were designed for chemicals, not microorganisms. Clear guidance for microorganisms is needed.

p. 45557Request for comment on economic impact. If the proposed rule were simplified, it would not be burdensome. As proposed, the rule will potentially enable only high value microorganisms to be commercialized; microorganisms of limited use, temporally and spatially, are unlikely to be developed. Organisms only used for research purposes, e.g. tracking of markers and traits in the environment are not likely to be processed because the time, effort and costs for such studies as proposed, are onerous.

We believe the proposed rule will increase the burden of those engaged in biotechnology research and its applications and impact negatively on this country's international competitiveness. Although the fees for submission are reasonable and the proposed rules are an improvement over the current PMN process, there is concern for the timeliness of EPA reviews of submissions and EPA decision-making. Long delays will significantly hinder biotechnology advancement, discourage additional R&D, and negatively impact international competitiveness. The burden of these proposed regulations should be compared to the existing PMN system, and to regulatory standards and requirements of other countries that are actively engaged in biotechnology and its commericalization. In an era of rapidly increasing global competitiveness, excessive domestic regulation will hinder biotechnology advancement and international competitiveness.

p. 45559Request for comment on regulatory flexibility analysis. The rationale can be questioned. The rule is based on conjecture. The mechanisms outlined will not lessen impacts on small business relative to the use of unmodified organisms that may provide the same function.

p. 45559Paperwork Reduction Act.It is not clear if the information required is only for PMN and SNUR. It is difficult to consider "473 hours per response" as a reasonable time input for proposing R&D experiments before MCAN. Comparable time development for experiments using unmodified organisms would be about 1 percent of this time.

p. 45562It is not clear to what extent health and safety studies need to be conducted. If the examples cited are equivalent to the requirements, they are onerous and open-ended in the aggregate compared to requirements with non-modified microorganisms.

p. 45568

It is not clear who pays the cost of EPA inspections and under what conditions inspection will be done; or if inspections can be performed under the auspices of another agency, e.g. NIH.

p. 45572725.155 Information Included in MCAN (c)(3)(ii) and (iv)These issues present technical difficulties. They are burdensome and EPA should not request this level of testing unless there is convincing reason for concern. For example, studies to address issues such as involvement in biogeochemical mineral or nutrient cycling (725.155) or studies to address ecological issues (725.3 as described on p. 45562) such as environmental effects on invertebrates, fish and plants (including acute and chronic toxicity tests, critical life stage tests, behavioral tests, algal growth tests, seed germination tests, plant growth and damage tests and microbial function tests) would require great expenditures of time, effort and money. In addition, the results would be open to question because there is a lack of acceptable and proven methodology in these areas.

p. 45576Under exemption conditions. The language should include provision for monitoring; this should be done because there are scientists who believe that even rhizobia are potentially a concern in the environment and rhizobia are known to establish themselves in soil and root environments.

p. 45577

TERA information. The requirements for reporting on (iii) on the test site and (iv) target organism are so extensive and open-ended that the cost and time to obtain such information are unlikely to offset the investment in research. They are not necessarily needed and may not provide any relevant information on the safety to humans and the environment. Many of these features would be temporal in any case.

In (2), the request for monitoring should require the submitter to describe the type, frequency and duration of monitoring. Such data will be essential for commercialization and valuable for subsequent alteration of policies and procedures under TSCA.

p. 45579(725.421)(b) The term, 'well characterized' is useful, but rather strong, since precise functions of many gene products (proteins) are not known or difficult to ascertain. This does not necessarily present a potential hazard. The word, "function" (b-1) could be replaced with "nature."

p. 45580725.422 - (d) Inactivation Procedures."The inactivation procedures must reduce microbial concentrations by at least 6 logs in liquid and solid wastes." This is too lenient. The intention was probably to have a 6 log overkill capacity built into an inactivation procedure. For example, if research is being conducted to optimize expression and cells are processed in some inactivating manner, it is probable that the starting concentration of cells will substantially exceed 106/mL. As a general rule 1 OD unit of E. coli is about 108 cfu/mL and in a substantially grown fermentation 109 to 1010 is not unusual. A six log reduction in such a population is insufficient.

725.422 - (e) Document Effectiveness."Provide and document effectiveness of features to reduce microbial concentration by at least 2 logs in aerosols and exhaust gases released from the structure." This may not be enough. For example, if you start with 106 bacterial cells in an aerosol and reduce it by 2 logs, this reduction would be insufficient. It is recommended that gases be vented through an integrity tested HEPA equivalent filter or be incinerated.

These containment issues should be closely coordinated and made complementary to the NIH Guidelines on containment which are widely understood and practiced.

Other Comments

We are of the opinion that regulatory regions should not be exempted from oversight. The locus of insertion can have unforeseen effects. However, it is the product that should be examined and not the process.

Synthetic sequences should be addressed. Sequences can be synthesized and inserted into a recipient. Depending on the nature and behavior of the construct and the recipient, they may be appropriate for oversight. Finally, sequences not known to occur in nature should be addressed. It may be prudent to subject such "unnatural" sequences to greater scrutiny than naturally occurring ones, at least initially.

The ASM appreciates the opportunity to comment on the proposed rule for microbial products of biotechnology. In preparing these comments, the ASM has consulted widely with expert scientists who are members of its committees on culture collections and environmental, agricultural, industrial, genetic and medical microbiology under the ASM's Public and Scientific Affairs Board. The ASM would be pleased to answer any questions, provide additional information or assist the EPA in the development of a workable framework for regulating microbial products of biotechnology.